In the previous project period, we performed an extensive analysis of the 8p11-p12 amplicon in human breast cancer cell lines and tissues to identify and validate novel breast cancer oncogenes. Using statistical analysis of copy number increase and over expression, we identified a subset of 21 genes as candidate oncogenes. Next, we directly tested the transforming function of these genes in human mammary epithelial cells. From these experiments, we identified four genes that are potently transforming in MCF-10A cells and three other genes with more modest transforming function. The most potently transforming genes identified, which include DDHD2, SPFH2, LSM1 and WHSC1L1, induce growth factor independent proliferation, anchorage-independent growth, invasive capacity, and altered morphogenesis in Matrigel. In addition, we identified gene combinations that effect the expression of transformed phenotypes. In the next phase of this work, we will perform experiments to understand the mechanistic basis for the transforming potential of these oncogenes, and we will examine their transforming function in human breast cancer cells in vitro, and in human and mouse mammary epithelial cells in vivo. The specific aims of the work in the next project period are: 1) To determine if the seven genes that induce transformed phenotypes in MCF-10A cells are directly transforming, and to determine if transformation is a common or a rare event in cells over expressing the oncogene;2) To determine if the genes from the 8p11 region that are amplified and over expressed in human breast cancer cell lines are required for growth and survival of these breast cancer cells compared with normal mammary epithelial cells or breast cancer cells without the amplicon. We will also test the hypothesis that some oncogenes on the 8p11 amplicon cooperate to influence the transformed growth potential of human breast cancer cells;3) To determine the influence of 8p11 oncogene over expression in the in vivo growth potential of human mammary epithelial cells, and to determine if these oncogenes can transform mouse mammary epithelial cells in vivo;4) To test the hypothesis that over expression of the short isoform of WHSC1L1 alters the histone methylation code and gene expression profile, resulting in cells that exhibit properties of tumor initiating cells, including enhanced self-renewal capacity, expression of markers of cancer stem cells, and ability to form mammospheres in culture. We will also test the hypothesis that induction of these altered phenotypes requires an intact PWWP domain and does not require the SET domain of the protein. It is essential to demonstrate unequivocally that newly discovered 8p11 transforming genes are bone fide breast cancer oncogenes, and to elucidate the mechanism by which they induce cell transformation in order to develop therapeutic strategies that target these oncogenes. PUBLIC HEALTH RELEVANCE: This project is aimed at developing a mechanistic understanding of newly discovered breast cancer oncogenes. New targeted drugs against cancer are most effective clinically when they attack the products of oncogenes that are responsible for cancer development. In order to develop new therapeutic strategies against breast cancer, it is essential that we clearly identify the oncogenes that drive the disease in different patients, and develop therapeutic strategies to more effectively treat patients with breast cancer. The work in this grant is directly relevant to the development of new targeted drugs against breast cancer.